LEZ 2 Biocompatibility Evaluation

8/9/2019 LEZ 2 Biocompatibility Evaluation

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Methods for evaluation of

BIOCOMPATIBILITY


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BIOMATERIALS

Biomaterials are defined as:

any substance (different from drugs) or combination of substances,with synthetic or natural origin, which can be used for any period oftime, as a whole or as a part of a system which treats, improves, or

replaces any tissue, organ, or function of the body

Biomaterial’s Science is:

The study and knowledge of interactions between non-living and livingmaterials

2nd Consensus Conference on Biomaterials Chester (UK), 7-8September 1991


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BIOMATERIAL’S SCIENCE


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Material Responces• Sweeling and Leaching

• Corrosion and Dissolution

• Friction and Wear

• Deformation and Failure

• Effect of surface morphology

• Effect of degradation products

Host Responces

Short term• Acute toxicity

• Irritation

• Sensitization

• Hemolysis

• Thrombogenicity

Long term

•

Subchronic and Chronic toxicity• Genotoxicity

• Carcinogenicity

• Effect on reproduction including

teratogenicity


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The Reality

• The host response, involving both humoral and cellular components isextremely complex

• Several of these components involve amplification or cascade events

• There is often a two-way relationship between the material variable and the

host response e.g. a degradation process is pro-inflammatory and the products

of inflammation enhance the degradation process

• Mechanical stability influences the host response, and in many situations the

host response determines the stability (e.g. osseointegration)

• The host response is time dependent

• The host response is patient specific, depending on age, sex, health status /

concomitant disease, pharmacological status, lifestyle, etc.

• Biocompatibility is species specific - testing materials in young rats in

Liverpool may be of no relevance to senior citizens in Sydney.


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The evaluation of the biocompatibility of materials,

i. e. the evaluation of the suitability of materials foruse in implantable medical devices, has evolved overapproximately the last 50 years.

1989: International Standards Organisation concerning biological

evaluation of medical devices (ISO 10993) and currently operating


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The ISO 10993 set entails a series of standards forevaluating the biocompatibility of a medical device prior to

a clinical study.

List of the standards in the Biological evaluetion of medical devicesISO 10993 (EN 30993)ISO 10993-1:2009 Evaluation and testing

ISO 10993-2:2006 Animal welfare requirementsISO 10993-3:2003 Tests for genotoxicity, carcinogenicity andreproductive toxicityISO 10993-4:2002/Amd 1:2006 Selection of tests for interactionswith blood

ISO 10993-5:2009 Tests for in vitro cytotoxicityISO 10993-6:2007 Tests for local effects after implantationISO 10993-7:2008 Ethylene oxide sterilization residualsISO 10993-8:2001 Selection of reference materialsISO 10993-9:1999 Framework for identification and quantification of

potential degradation products


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ISO 10993-10:2010 Tests for irritation and delayed-typehypersensitivityISO 10993-11:2006 Tests for systemic toxicity

ISO 10993-12:2007 Sample preparation and reference materials(available in English only)ISO 10993-13:1998 Identification and quantification of degradationproducts from polymeric medical devicesISO Identification and quantification of degradation products from

ceramicsISO 10993-15:2000 Identification and quantification of degradationproducts from metals and alloysISO 10993-16:1997 Biological evaluation of medical devices Part 16:Toxico-kinetic study design for degradation products and leachablesISO 10993-17:2002 Establishment of allowable limits for leachablesubstancesISO 10993-18:2005 Chemical characterization of materialsISO/TS 10993-19:2006 Physico-chemical, morphological andtopographical characterization of materialsISO/TS 10993-20:2006 Principles and methods for immunotoxicology

testing of medical devices


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ISO 10993-5

Tests for Cytotoxicity-In vitro Methods

Indirect Method: Elution test

Direct Method: Contact test

In standard cytotoxicity test methods, cell monolayers are grown tonear confluence in flasks and are then exposed to test or control

materials directly or indirectly by means of fluid extracts.


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Indirect Method: Elution testIn the elution test method, extracts are obtained by placing the test

and control materials in separate cell culture media under standard

conditions. Each fluid extract obtained is then applied to a cultured-cell monolayer, replacing the medium that had nourished the cells to

that point. In this way, test cells are supplied with a fresh nutrient

medium containing extracts derived from the test material or control.

The cultures are then returned to the 37°C incubator and periodically

removed for microscopic examination at designated times for as long

as three days.


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Direct Method: Contact test

In the contact test method, samples of test and control material can be

applied directly to monolayers of cells covered with nutrient medium.

During the subsequent incubation period, extracts from the samples will

migrate into the nutrient medium or through the nutrient agar overlayto the underlying cells. After incubation, the monolayers are evaluated

in terms of the presence or absence of a zone of cellular effects

beneath and surrounding the sample. Extraction conditions in the direct

contact methods are less rigorous than in the elution test. However,

this method is particularly useful if only very small quantities of samples

are available or when only one surface of a material needs to be

evaluated.


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Indirect Method: Elution test Direct Method: Contact test


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Quantitative evaluation

Test for determination of cell viability:

Trypan blue dye exclusion assay

MTT or WST1 test

Test for determination of enzymatic release:

LDH assay

Cytotoxicity Evaluation


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Trypan blue dye exclusion test

Cell viability is determined by stainingthe cells with trypan blue. Trypan blueis a vital stain used to selectively

colour dead tissue or cells in blue. Livecells or tissues with intact cellmembrane are not coloured. They areexcluded from staining while dead

cells are shown as a distinctive bluecolour under a microscope.

Percentage % of viable cells =N° of unstained cells/total N° of cells x 100

Ex: 6/9=0,6660.66*100=66,6%Percentage of viable cells


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MTT or WST1 assay

The MTT assay is a colorimetricassay for measuring the activityof enzymes that reduce MTT orclose dyes (WSTs) to formazan,giving a purple color.

A main application of this assay allows to assess theviability and the proliferation of cells. It can also be usedto determine cytotoxicity of potential medicinal agentsand toxic materials, since those agents would stimulate or

inhibit cell viability and growth.

A microtiter plate after an MTTassay. Increasing amounts ofcells resulted in increased

purple colouring.

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LDH assayLDH is a cytoplasmic enzyme that is released into the

cytoplasm upon cell lysis. The LDH assay, therefore, is ameasure of membrane integrity and of cell viabilitybecause LDH release into the media is a marker of celldead.

(1) LDH oxidizeslactate to pyruvate

(2) Pyruvate reacts with the tetrazolium saltto form formazan

(3) the water-soluble formazan dye isdetected spectrophotometrically at awavelength of 340 nm


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Qualitative evaluation

Microscopy examination:

Cells are observed for visible signs of toxicity (such as a change in the

size or appearance of cellular components or a disruption in their

configuration) in response to the test and control materials (Figures 1

and 2).

Cytotoxicity Evaluation

Figure 1. A confluentmonolayer (100 x

magnification) of L929mouse fibroblast cells.

This appearance isindicative of a

noncytotoxic response inthe elution test method.

Figure 2. Cytotoxic reactionin the elution test method.

The L929 mouse fibroblastcells cells (100 xmagnification) are grainy

and lack normal cytoplasmicspace; the considerable openareas between cells indicate

that extensive cell lysis(disintegration) has

occurred.


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Morphological Evaluation of Cell Death

Necrosis vs ApoptosisEtiology of Cell Death

Necrosis:A pathological response to cellular injury. It is the sum of themorphologic changes that follow cell death in a living tissue or organ

Apoptosis:a physiological process that includes specific suicide signals leading tocell death

Major Factors

Accidental

NECROSIS

Genetic

APOPTOSIS


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Necrosis

Consequences of cell injury

NORMALREVERSIBLE CHANGESDisaggregated polysomesFocal chromatin marginationMild mitochondria swelling

IRREVERSIBLE CHANGESHigh amplitude mitochondria swelling

Mitochondria matrix densitiesProgressive dilatation of endoplasmic reticulumLysosomal rupturePlasma membrane ruptureNuclear dissolutionLoss of recognisable organelles


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Apoptosis

Morphological changes that occur during the apoptosis:

I) The normal cellII) Shrink and the condensed

chromatin collapses into

crescents around the nuclearenvelope

III)The membrane begins to bulgeand bleb

IV) The blebing increases and thecell finally breaks into a number

of apoptotic bodiesV) Which lyse in vitroVI) And phagocytoses in vivo


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Necrosis: a pathological responseto cellular injury

Apoptosis: a physiological response tospecific suicide signals, or lack of survivalsignals

Chromatin condenses and migrates tonuclear membrane. Internucleosomalcleavage leads to laddering of DNA at thenucleosomal repeat length, ca. 200 bp.

Cytoplasm shrinks without membranerupture

Blebbing of plasma and nuclear membranes

Cell contents are packaged in membranebounded bodies, internal organelles stillfunctioning, to be engulfed by neighbours

Epitopes appear on plasma membranemarking cell as a phagocytic target.

No spillage, no inflammation

Chromatin clumps

Mitochondria swell and rupture

Plasma membrane lyses

Cell contents spill out

General inflammatory response is

triggered


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ISO 10933-4

Selection of tests for interactions withblood-Hemocompatibility

Blood represents one of the most complex biochemical systems in living

organisms, and its various components play integral roles in several lifefunctions. Because these functions are critical, medical devices thatcontact blood during routine use must be hemocompatible, therefore,they must not adversely interact with any blood components so as tocause their inappropriate activation or even destruction.

Blood/device interaction:any interaction between blood or any component of blood and a deviceresulting in effects on the blood, or any organ or tissue or on thedevice. Such effects may or may not have clinically significant or

undesiderable consequences.


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Blood is a specialized fluid of body that delivers necessary substancesto the body's cells– such as nutrients and oxigen – and transports

waste products away from those same cells.

It is composed of a multitude

of cell types, suspended in aliquid called blood plasma.

The blood cells are mainly:Erythrocites-they transport oxigenLynphocytes-cells that destroy invading pathogens

Platelets-important cells in clotting of blood


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The types of tests required by ISO depend on the bloodcontact category and on the time of contact of the

device or material (< 24h, 24h > t < 30 days, > 30 days):

- Device that hasn’t contact with circulating blood(blood cell counter, etc..)- Device that has contact with circulating blood

indirectly• system to collect blood (blood bag, etc..)directly• external communicating devices (dreinagecatherters, buttarfly needles, etc..)

• implant devices (stents, cardiac valve, etc..)


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Test Methods Comments

Thrombosis Light microscopy (adhered platelets, leukocytes,

aggregates, erythrocytes,

fibrin, etc.)

Light microscopy can be replaced byscanning electron microscopy if thenature of the material presents technical

problems for light microscopy.Coagulation Partial thromboplastin

time (nonactivated)

Platelets Platelet count

Hematology Leukocyte count anddifferential; hemolysis(plasma hemoglobin)

Hemolysis is regarded as an especiallysignificant screening test to perform inthis category because of its measurementof red blood cell membrane fragility incontact with materials and devices. The

method used should be one of thenormative standard test methods forhemolysis.

Immunology C3a, C5a, TCC, Bb, iC3b,C4d, SC5b-9

A panel including the last four testsencompasses the various complementactivation pathways.


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Some examples of Tests

• Hemolysis (ASTM F 756)ASTM F 756 is a standard test method to evaluate whether direct contact withthe matherial or an extract of the material would cause in vitro red blood cellhemolysis. This test involves a quantitative measurement of plasma hemoglobinby using a hemoglobin reagent and spettrophotometric measurement atwavelenght of 540 nm. An increase in plasma hemoglobin correlates with lysis of

red blood cells, thereby indicating hemolytic activity of the material exposed tothe cells.• Coagulation (determination of the rate of clot formation)A device's effects on blood coagulation may be measured in vitro by determiningthe rate of clot formation or the partial thromboplastin time (PTT) of plasmaexposed to the biomaterial or device during an incubation period.• ThrombosisThrombosis may be addressed by performing either an in vivo or ex vivo test. Anevaluation of the thrombogenic potential of a device typically involves placingthe device in a simulated clinical setting for a period of time, then removing thedevice and evaluating the extent of thrombus formation on or in it.


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• Platelets (platelets count, adhesionand aggregation)PLATELETS COUNT Manually platelets count

Automatic cell counterADHESION and AGGREGATIONSEM analysis


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ISO 10993-3

Tests for genotoxicity, carcinogenicity andreproductive toxicity

• Gene Mutations

• Chromosomal Aberrations

• DNA Effects

• Gene Mutation Tests

• Chromosomal Aberration Tests

• DNA Effect Tests

Three major type of

genotoxic effect:

Three major type of tests for

genotoxicity, carcinogenicity andreproductive toxicity :


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Gene Mutation test:

Ames TestThe Ames bacterial reverse mutationassay is most commonly used to detect

gene mutations and utilizes histidine-dependent Salmonella typhimurium strainsas the test organisms. Rat liver extractare incorporated into a portion of the testorganisms to simulate whole-animalexposure. Following exposure to the fluid

extract from the test material, theorganisms are plated in triplicate ontohistidine-free growth nutrient agar andincubated for a specified period. Thecolonies are then enumerated and thesedata are compared to counts obtained for

negative control conditions. Since theunreverted test strains will not growwithout histidine, any further growthindicates that exposure to a genotoxicagent has caused point mutations thathave produced bacterial strains that no

longer require histidine.

• Test system – auxotrophic strain of


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Ames test

His–

bacteria

Dies in anormalmedium

Mediumcontaininghistidine

Normalmedium

His+

bacteriaReverse mutation

Mutagen

• Test system – auxotrophic strain ofSalmonella typhimurium – survives only inmedium with histidine (dies in normalmedium without histidine)• After treatment with mutagen some

auxotrophic cells are turned into normalones that synthesize histidine and survive ina normal medium.• These cells are called revertants (due toreverse mutation).


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0 Negativecontrol

Positivecontrol

A dish with acompound to

be tested

GENOTOXICITY

CONFIRMED

SPONTANEOUSREVERTANTS

IS USED FOR

CONTROL OF

THE TEST

Result of Ames test


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Chromosomal aberration testing detect chromosomaldamage induced after one cellular division; structuralchanges in the chromosomes are evaluated while cellsare in the metaphase stage of division. The in vitromodel employs Chinese hamster ovary cells. Gaps,breaks, and exchanges are other examples ofobservable aberrations (Figure).

ChromosomalAberration Tests

The mouse bone marrow micronucleus test is an in vivo assay that detectdamage to the chromosomes or the mitotic apparatus of immature red bloodcells found in bone marrow.

During cell division, if the chromosomes are broken or themitotic apparatus of the cell is damaged, chromosomefragments may be incorporated in secondary nuclei

instead of into the main nucleus. Secondary nuclei aremuch smaller than the main nucleus and are referred toas micronuclei.

When erythroblasts develop into polychromatic erythrocytes (PCEs), the mainnucleus is extruded but any micronuclei that are present remain behind. Thus,

an increase in the number of micronucleated PCEs in animals treated with thetest article extract is an indication of the presence of a genotoxin (Figure).

DNA Effects Tests


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Possible results of

chromosomal analysisPercentage of cells with

chromosomal aberrationsResult

Less then 2%Normal finding, spontaneous

level of aberrations

2 –

4 %

A border result – mutagenic

effect is neither confirmednor excluded

More than 4%Mutagenic effect confirmed

with high probability


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Result of micronucleus test


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Carcinogenicity Test

Carcinogenicity:The potential of a device material that comes in contact with a patientto cause or incite the growth of malignant cells.

The ISO 10993 standards, which covers genotoxicity, carcinogenicity,

and reproductive toxicity, describes carcinogenicity testing as themeans "to determine the tumorigenic potential of devices, materials,and/or extracts to either a single or multiple exposures over a periodof the total life-span of the test animal“. Specifically, such testingshould be considered for a device that will have permanent contact(longer than 30 days) with tissues. The standard further indicatesthat "carcinogenicity tests should be conducted only if there aresuggestive data from other sources." Thus, not every device needs tobe subjected to this time-consuming (total life-span) and expensivetesting (in vivo test).


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In vivo is experimentation using a whole, living organism as opposed toan in vitro (i.e., in a test tube or petri dish) controlled environment.

Animal tests and clinical trials are two forms of in vivo research.

In vivo Test

Supporters of the use of animals in experiments argue that virtuallyevery medical achievement in the 20th century relied on the use ofanimals in some way, arguing that even sophisticated computers areunable to model interactions between molecules, cells, tissues, organs,organisms, and the environment, making animal research necessary in

many areas.Other scientists arguing that it is cruel, poorly regulated, that medicalprogress is being held back by misleading animal models, that some ofthe tests are outdated, that it cannot reliably predict effects inhumans, that the costs outweigh the benefits, or that animals have an

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LEZ 2 Biocompatibility Evaluation